Bottom Line:
Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACTThe recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

Mentions:
Antisense knockdown using morpholinos targeting either the elys 5′ UTR, translation initiation codon or the exon 30 splice acceptor (Figure S4) phenocopied the flo retinal (Figure 2D–2F and 2U) and tectal defects (Figure 2G–2I). Phenocopy was present in only a minority of embryos injected with single morpholinos (10%–12%), most likely because of the high levels of maternal elys mRNA relative to the levels of zygotically derived mRNA (Figure 3A). Combined injection of the 5′-UTR and exon 30 splice donor morpholinos led to phenocopy in 30% to 50% of injected embryos (n = >300 injected embryos). All elys-morpholino injected larvae with retinal and tectal apoptosis had minimal exocrine pancreas tissue (Figure 2S–2T) as do flo larvae [25]. Similarly, like flo mutants [4], all of the affected morpholino injected larvae lacked intestinal goblet cells (Figure 2J–2L) and had a dramatic reduction in non-enteroendocrine secretory cells and enterocytes (Figure 2M–2R) as revealed by previously described monoclonal antibodies [32]. Apoptosis, which is a prominent feature of the flo intestinal phenotype (discussed below), was not evident in the intestine of the elys-morpholino injected larvae despite clear evidence of NPC disruption. Although partial intestinal phenocopy by the Elys knockdown was not unexpected because morpholino knockdowns are often transient, these data raise the possibility that apoptosis caused by loss of Elys function may occur independently of altered NPC assembly seen in flo mutants.

Mentions:
Antisense knockdown using morpholinos targeting either the elys 5′ UTR, translation initiation codon or the exon 30 splice acceptor (Figure S4) phenocopied the flo retinal (Figure 2D–2F and 2U) and tectal defects (Figure 2G–2I). Phenocopy was present in only a minority of embryos injected with single morpholinos (10%–12%), most likely because of the high levels of maternal elys mRNA relative to the levels of zygotically derived mRNA (Figure 3A). Combined injection of the 5′-UTR and exon 30 splice donor morpholinos led to phenocopy in 30% to 50% of injected embryos (n = >300 injected embryos). All elys-morpholino injected larvae with retinal and tectal apoptosis had minimal exocrine pancreas tissue (Figure 2S–2T) as do flo larvae [25]. Similarly, like flo mutants [4], all of the affected morpholino injected larvae lacked intestinal goblet cells (Figure 2J–2L) and had a dramatic reduction in non-enteroendocrine secretory cells and enterocytes (Figure 2M–2R) as revealed by previously described monoclonal antibodies [32]. Apoptosis, which is a prominent feature of the flo intestinal phenotype (discussed below), was not evident in the intestine of the elys-morpholino injected larvae despite clear evidence of NPC disruption. Although partial intestinal phenocopy by the Elys knockdown was not unexpected because morpholino knockdowns are often transient, these data raise the possibility that apoptosis caused by loss of Elys function may occur independently of altered NPC assembly seen in flo mutants.

Bottom Line:
Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

Affiliation:
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACTThe recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.